Document text
Principal Investigator: Kui Kiu Chan
Organization: ORTHOGONAL BIOLOGICS, INC.
Fiscal Year: 2021
Award: $95,981
Funding agency: National Institute of Allergy and Infectious Diseases
ABSTRACT
The spike protein (S) of SARS coronavirus 2, the pathogen responsible for COVID-19, binds angiotensin-
converting enzyme 2 (ACE2) as an entry receptor, triggering conformational changes in S that drive fusion of
the viral envelope and host cell membrane. Infection is inhibited by neutralizing antibodies that block the
ACE2-binding site on S, yet escape mutations in S rapidly emerge towards monoclonal antibodies in tissue
culture. Furthermore, monoclonal antibodies are generally strain specific, and many do not recognize with high
affinity both human SARS-CoV-1 and SARS-CoV-2, yet alone exotic bat betacoronaviruses that are a reservoir
for future outbreaks. As an alternative for biologic drug development, we have used deep mutagenesis to
guide the engineering of an exceptionally broad soluble decoy receptor that binds with tight picomolar/low-
nanomolar affinity to S from all bat and human SARS-associated coronaviruses tested. The engineered decoy
potently neutralizes authentic SARS-CoV-1 and SARS-CoV-2 with an efficacy that rivals monoclonals under
commercial development, and has desirable properties for manufacture at scale. The engineered decoy also
catalytically converts angiotensin II to vasodilatory peptide products that might directly address symptoms of
COVID-19, providing us with a unique potential therapeutic that has dual mechanisms of action. Our proposal
investigates whether the SARS-CoV-2 spike can mutate to escape neutralization by the engineered decoy
receptor, and addresses final optimization of the engineered protein as an IgG1-Fc fusion before advancement
to an IND-enabling program. For SARS-CoV-2 to become resistant to the engineered decoy, mutations in S
must decrease affinity to the decoy while maintaining binding to human ACE2 receptors. To identify such S
variants, we have used saturation mutagenesis of the receptor-binding domain coupled with a selection for
tight binding to wild type ACE2 in the presence of competing soluble decoy. Following deep sequencing, a
small number of mutations were found to be enriched, but it is unclear whether any of these mutations do
indeed preferentially bind wild type ACE2 and if so, to what degree they have achieved specificity. Based on
this preliminary data, (Aim 1) we will validate whether mutations in S can be found that discriminate between
human ACE2 and the engineered decoy, and characterize the variants for their affinities and expression levels.
Thus far, we can conclude that possible resistance mutations appear to be very rare and generally require
more than one nucleotide change within a codon, but further quantitative characterization is needed.
Simultaneously, (Aim 2) we will rapidly optimize fusions of the engineered decoy with the Fc region of IgG1 for
enhanced serum stability. Our current IgG1-fusion construct (which was based on rational, structure-guided
design) is highly expressed, stable and binds SARS-CoV-2 S with picomolar affinity. We will finalize
optimization of the protein by scanning suitable fusion sites between the engineered decoy and IgG1,
assessing protein quality by activity, stability and expression.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><ACE2><ARDS><Acute Respiratory Distress><Acute Respiratory Distress Syndrome><Address><Adrenal Cortex Hormones><Adult ARDS><Adult RDS><Adult Respiratory Distress Syndrome><Affinity><Amino Acids><AngII><Angiotensin II><Angiotensins><Animal Model><Animal Models and Related Studies><Animals><Antigenic Determinants><Associated Viruses><Bats><Binding><Binding Determinants><Binding Sites><Biological><Biotech><Biotechnology><Blocking Antibodies><Blood Serum><COVID crisis><COVID epidemic><COVID pandemic><COVID-19><COVID-19 crisis><COVID-19 epidemic><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 infection><COVID-19 pandemic><COVID-19 public health crisis><COVID-19 therapy><COVID-19 treatment><COVID-19 virus><COVID19><COVID19 crisis><COVID19 epidemic><COVID19 global health crisis><COVID19 global pandemic><COVID19 health crisis><COVID19 infection><COVID19 pandemic><COVID19 public health crisis><COVID19 therapy><COVID19 treatment><COVID19 virus><CV-19><CV19><Cell Body><Cell membrane><Cells><Chimera Protein><Chimeric Proteins><Chiroptera><Clinic><Clinical><Clinical Treatment Moab><CoV-2><CoV2><Codon><Codon Nucleotides><Combining Site><Competitive Binding><Complex><Coronaviridae><Coronavirus><Corticoids><Corticosteroids><Coupled><Cytoplasmic Membrane><DNA Recombination><Da Nang Lung><Data><Development><Dexamethasone><Disease><Disease Outbreaks><Disorder><Drug Targeting><Drugs><Engineering><Epitopes><Escape Mutant><FDA approved><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Freeze Drying><Freeze Dryings><Freezing><Fusion Protein><Future><GS-5734><Genetic Alteration><Genetic Change><Genetic Differentiation><Genetic Divergence><Genetic Drift><Genetic Recombination><Genetic defect><Genetics-Mutagenesis><HCoV><Half-Life><Heart><Human><IgG1><Illinois><In Vitro><Infection><Inflammation><Investigational Drugs><Investigational New Drugs><Libraries><Lung><Lung Inflammation><Lung Respiratory System><Lung damage><Lyophilization><Measurement><Medical><Medication><Mice><Mice Mammals><Modern Man><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Monoclonal Antibodies><Murine><Mus><Mutagenesis><Mutagenesis Molecular Biology><Mutate><Mutation><Noise><Nucleotides><Outbreaks><Patients><Peptide Hormone Gene><Peptides><Pharmaceutic Preparations><Pharmaceutical Preparations><Plasma Membrane><Polymerase><Position><Positioning Attribute><Property><Protein Engineering><Proteins><Publishing><Reactive Site><Receptor Protein><Recombination><Resistance><SARS><SARS Virus><SARS corona virus><SARS corona virus 2><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV disease><SARS-CoV-2><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 infection><SARS-CoV-2 pandemic><SARS-CoV-2 therapy><SARS-CoV-2 treatment><SARS-CoV2><SARS-CoV2 epidemic><SARS-CoV2 infection><SARS-CoV2 pandemic><SARS-Related Coronavirus><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Safety><Satellite Viruses><Scanning><Serum><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 pandemic><Severe acute respiratory syndrome related corona virus 2><Shock Lung><Site><Source><Specificity><Stiff lung><Structure><Symptoms><Temperature><Testing><Therapeutic><Toxicology><Universities><Variant><Variation><Veklury><Vendor><Viral><Virus><Virus Replication><Wuhan coronavirus><aminoacid><angiotensin converting enzyme 2><angiotensin converting enzyme II><base><beta CoV><beta coronavirus><betaCoV><betacoronavirus><commercialization><competitively bound><conformation><conformational state><corona virus><corona virus disease 2019><corona virus disease 2019 epidemic><corona virus disease 2019 pandemic><coronavirus disease 2019><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 infection><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease 2019 therapy><coronavirus disease 2019 treatment><coronavirus disease 2019 virus><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><deep sequencing><design><designing><developmental><drug development><drug/agent><elderly patient><flow cytophotometry><genetic protein engineering><genome mutation><hCoV19><human CoV><human corona virus><human coronavirus><immunogenicity><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><lead candidate><lead optimization><lung injury><mAbs><model of animal><model organism><mutant><nCoV2><nano-molar><nanomolar><neutralizing antibody><neutralizing mAb><neutralizing monoclonal antibodies><nucleoside analog><older patient><pandemic><pandemic disease><pathogen><peptide hormone><physical property><plasmalemma><prevent><preventing><programs><protective effect><protein design><pulmonary><receptor><receptor binding><receptor bound><remdesivir><resistance mutation><resistant><resistant mutation><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><severe acute respiratory syndrome coronavirus 2 therapy><severe acute respiratory syndrome coronavirus 2 treatment><severe acute respiratory syndrome-CoV><stability testing><tissue culture><treat COVID-19><treat COVID19><treat SARS-CoV-2><treat coronavirus disease 2019><treat severe acute respiratory syndrome coronavirus 2><viral multiplication><viral replication><virus multiplication><wet lung><zoonotic CoV><zoonotic coronavirus><β CoV><β coronavirus><βCoV>